ABSTRACT Rhabdomyosarcoma (RMS) is a heterogeneous collection of cancers demonstrating varying degrees of skeletal muscle differentiation. Although accounting for ~8% of pediatric malignant solid tumors, RMS is the most common soft tissue sarcoma in children younger than 14 years. The two major histologic subtypes of RMS are embryonal (eRMS) and alveolar (aRMS). High risk patients have a 5-year survival of 30%, and outcome is very poor for children whose tumors express the PAX3-FKHR fusion gene; when metastatic, their 5-year survival is <8%. This signature genetic change is found only in aRMS and considered a tumor-specific oncogene, but has no molecularly targeted treatment. To address gaps in knowledge of RMS, we have created a new model for this disease based on the conversion of primary human skeletal muscle cells to their tumorigenic counterpart, using a defined set of genetic changes. Using this model, we found that human skeletal muscle myoblasts may be converted to cells that generate tumors mimicking RMS when tested as xenografts in immunodeficient mice. Having established that primary human cells of skeletal muscle origin can give rise to RMS, we studied the repercussions of expressing PAX3-FKHR in them, and discovered two phenotypes that may underlie its oncogenic behavior. First, when PAX3-FKHR was stably expressed as an early genetic change, it enabled bypass of the senescence checkpoint and served as an initiating oncogenic hit for the development of skeletal muscle tumors. Second, when PAX3-FKHR was stably expressed as a late genetic change, it shortened the latency of in vivo tumor formation from 11 to 2 weeks, possibly through activation of the Ras pathway, since in control experiments PAX3-FKHR could functionally substitute for the RAS oncogene. In this proposal, we wish to understand how PAX3-FKHR enables bypass of the senescence checkpoint, and how it accelerates tumorigenesis in previously transformed cells. To accomplish this, we will (1) examine candidate proteins that are downstream of PAX3-FKHR for their role in overcoming the senescence checkpoint, using both gain-of-function and loss-of-function approaches, and (2) examine the accelerated tumor cells for enhanced self-sufficiency in growth signaling, apoptosis, and/or angiogenesis, and the role of the Ras pathway in this PAX3-FKHR-augmented tumorigenesis. The accomplishment of these aims will provide insight into the genesis of this pediatric malignancy, and provide new therapeutic targets for study. In addition, this genetically defined model will serve as a template for the systematic investigation of other human sarcomas.